This invention claims priority of the German patent application DE 100 37 783.1 which is incorporated by reference herein.
The invention concerns a method for phase correction of position and detection signals in scanning microscopy. The scanning microscope can also be configured as a confocal microscope.
The invention furthermore concerns an apparatus for phase correction of position and detection signals in scanning microscopy.
The invention moreover concerns a scanning microscope that makes possible the phase correction of position and detection signals.
In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted by the sample. The focus of the illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture stop (called the xe2x80x9cexcitation stopxe2x80x9d), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected light or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen passes by way of the beam deflection device back to the beam splitter, and passes through the latter in order then to be focused onto the detection stop, behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by sensing image data in layers.
Ideally, the track of the scanning light beam on or in the specimen describes a meander, i.e. one line is scanned in the X direction with a constant Y position, then X scanning is halted and a Y displacement is used to pivot to the next line to be scanned, and that line is then scanned in the negative X direction with a constant Y position, etc.
The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus sampled one scan point at a time. The reading must be unequivocally associated with the pertinent scan position so that an image can be generated from the measurement data. Advantageously, for this purpose the status data of the adjusting elements of the beam deflection device are also continuously measured, or (although this is less accurate) the reference control data of the beam deflection device are used directly.
A precise association of the position signals with the detection signals is particularly important. Transit time differences and the differing processing times of the detectors sensing the signals must be taken into account in the association, for example using delay elements. Very stringent requirements in terms of stability must be applied here: for an image width of 1000 image points, for example, the transit time fluctuations must remain well below 0.1%.
As scanning speed increases, the scanned track deviates more and more from a meander shape. This phenomenon is attributable essentially to the inertia of the moving elements. With rapid scanning, the scanned track is more similar to a sine curve, but it often happens that the trajectory portion for the scan in the positive X direction differs from the trajectory portion when scanning in the negative X direction.
Even if electronic elements are provided to compensate for transit time differences and processing times, a compensation is still performed only upon manufacture of the scanning microscope. Strictly speaking, however, this setting applies to only one scanning speed.
Unfortunately both the signal-carrying elements and the electronic components that are used for compensation, as well as the detectors sensing the reading, are temperature-sensitive, so that association errors occur if the temperature is even slightly variable.
In addition to the problem that compensation is valid for only one scanning speed, an additional complication is the fact that it is valid only for the temperature prevailing at the time the scanning microscope was manufactured.
The compensation errors are evident in particularly disruptive fashion in the context of meander-shaped scanning: the compensation errors for scanning in the positive X direction and for scanning in the negative X directions act in opposite directions, resulting in images with pronounced comb-like distortion.
It is therefore the object of the invention to describe a method for scanning microscopic preparations with a light beam in which an optimum association of position signals and detection signals is made possible even with changing scan parameters and environmental parameters.
The aforesaid object is achieved by a method comprising the following steps:
generating a position signal from the position of a beam deflection device and generation, from the light proceeding from the specimen, of at least one detection signal pertinent to the position signal;
transferring the position signal and detection signal to a processing unit;
determining a correction value; and
transferring the correction value to the processing unit to compensate for time differences between the position signal and detection signal.
An additional object of the invention is an apparatus for phase correction of position signals and detection signals in scanning microscopy, with which an optimum association of position signals and detection signals is made possible even with changing scan parameters and environmental parameters.
This object is achieved by means of an apparatus which comprises:
means for generating a position signal from the position of a beam deflection device, and means for generating, from the light proceeding from the specimen, a detection signal pertinent to the position signal, are provided;
a processing unit receiving the position signal and the detection signal; and
means for determining a correction value, which are connected to a processing unit and which transfer to the processing unit the correction value to compensate for time differences between the position signal and detection signal, are provided.
A further object of the invention is to create a scanning microscope which is correspondingly configured to achieve a phase correction of position signals and detection signals of a scanning microscope, an optimum association of position signals and detection signals being made possible even with changing scan parameters and environmental parameters.
This object is achieved by a scanning microscope which comprises: an illumination system for generating a light beam; a scanning module for scanning the light beam over a specimen; at least one detector that receives light proceeding from the specimen; means for generating a position signal from the position of the scanning module; means for generating, from the light proceeding from the specimen, a detection signal pertinent to the position signal; a processing unit receiving the position signal and the detection signal; and means for determining a correction value, which are connected to the processing unit and which transfer to the processing unit the correction value to compensate for time differences between the position signal and detection signal.
The invention has the advantage of proposing to the user several possibilities allowing easy correction of the time difference between the position signal and detection signal. In an advantageous embodiment, adjustment of the time difference is performed directly by the user. For that purpose, sliders are displayed on one of the displays, and adjustment is accomplished by the fact that the user modifies the position of the slider, for example with the mouse, in such a way that a time difference no longer occurs between the position signal and detection signal. The result of the new setting is displayed on the display in real time. For example, a sharp image is visible on the display when the position signal and detection signal are imaged in phase or appropriately corrected.
Automatic setting of the time difference is also possible. A control circuit having a temperature sensor and a data storage unit is provided for this purpose. Correction values pertinent to each scanning speed and temperature, which were ascertained in a series of calibration measurements, are stored in the data storage unit. The temperature is measured continuously or at fixed time intervals, and the correction value pertinent to the measured temperature and the present scanning speed is read out from the data storage unit and the elements for compensating for transit time differences and measured value processing times are set. The correction values can be stored, for example, in the form of a table.
A further embodiment for automatic setting of the transit time compensation comprises a feedback control loop with an image processing software programxe2x80x94as also known, for example, from autofocus systemsxe2x80x94which ascertains, from the present image of the specimen, the optimum setting of the transit time compensation in terms of optimum image sharpness. This could be accomplished, for example, by a correlation comparison of the detection signals of adjacent lines.